Unit 4: Human Proteins, Organ Failure, and Transplants - Comprehensive Notes

Unit 4 Cram Sheet Notes

Introduction

  • Unit 4 introduces Diana Jones, Judy Smith's sister, who is a Type II Diabetic.
  • Diana manages her diabetes with an insulin pump.
  • Her past choices significantly impact her future health.

Lesson 4.1: Manufacturing Human Proteins

  • The unit begins with the study of insulin, a crucial hormone for diabetics.
  • Insulin's Role: Insulin regulates blood sugar levels.
  • Diabetes: In some forms of diabetes the body does not produce insulin, causing sugar to remain in the bloodstream and damage tissues.
  • Diabetics who cannot produce insulin require external insulin sources.
  • Historical Insulin Sources: Previously, insulin was extracted from cow pancreases.
  • Cow insulin was less effective than human insulin.
  • Modern Insulin Production: Human insulin is now produced using bacteria.
  • This production involves bacterial transformation and recombinant DNA technology.
Bacterial Transformation
  • Process Overview: Modifying bacteria to produce specific proteins.
  • Starting Point: Using bacteria to "grow" the desired protein.
  • Recombinant DNA Technology: Custom-designing bacteria to produce specific proteins.
  • Plasmids:
    • Bacteria contain plasmids: small, circular DNA strands.
    • Plasmids replicate with bacteria and often carry antibiotic resistance genes.
    • Plasmids can be modified through genetic engineering to carry new traits.
  • Plasmid Modification Steps:
    • 1. Sequencing: Plasmids are sequenced to determine their genetic composition.
    • 2. Restriction Enzymes: Restriction enzymes are used to cut the plasmid DNA at specific sequences.
    • 3. Cutting: Restriction enzymes cut plasmid DNA, creating "sticky ends" with unpaired nucleotides.
    • 4. Gene Insertion: The desired gene fragment is mixed with the cut plasmids.
    • 5. Binding: Sticky ends of the plasmid and the gene fragment bind together.
    • 6. DNA Ligase: DNA ligase seals the bonds between the plasmid and the gene fragment, creating a new plasmid containing the desired gene.
    • The additional DNA contains the gene for the protein the bacteria will produce.
  • Modified plasmids are introduced into bacteria.
  • Bacterial Selection:
    • E. coli is a preferred bacterium due to its non-infectious nature.
    • E. coli is mixed with the modified plasmid and subjected to shock.
  • Calcium Chloride Treatment: Bacteria are chilled in calcium chloride which neutralizes the negative charge of DNA phosphate and phospholipids in cell membrane, enabling plasmids to enter cells.
  • Heat Shock: Bacteria undergo heat shock, increasing cell membrane permeability for plasmid entry.
  • Recovery: Bacteria recover in nutrient broth, sealing plasmids inside.
  • Plating: Transformed bacterial cells are plated on agar containing a specific antibiotic.
  • Selection: Antibiotic resistance gene in the plasmid ensures only transformed bacteria grow.
Protein Purification
  • Goal: Extracting human proteins from bacteria.
  • Process:
    • 1. Bacteria containing the human protein are grown on a petri dish since the gene codes for both the human protein and antibiotic resistance.
    • 2. A single colony is transferred to a small vial and grown overnight.
    • 3. The culture is centrifuged, forming a bacterial pellet.
    • 4. The pellet is resuspended in lysozyme, which ruptures cell membranes.
    • 5. The solution is recentrifuged to separate cell waste from proteins.
    • 6. Binding Buffer: A binding buffer is added that binds to the proteins in solution.
    • 7. Chromatography Column (First Run): The fluid is run through a chromatography column; all proteins bind to pellets within the column.
    • 8. Salty Buffer Wash: A slightly salty buffer removes unwanted (hydrophilic) proteins, leaving desired (hydrophobic) proteins.
    • 9. Low-Salt Buffer Wash: A low-salt buffer separates the desired hydrophobic proteins (e.g., GFP) from the column into a collection tube.
  • Note: This process is specific to hydrophobic proteins.
  • Final Step: Verify product quality and purity before distribution.
Diagram of Protein Purification
  • Starting Point: Bacterial colonies transformed with pGLO plasmid DNA.
  • Day 1:
    • Pick a single fluorescent green colony from the agar plate using a sterile inoculation loop.
    • Inoculate into nutrient broth containing ampicillin and arabinose.
    • Grow overnight at 32°C or 2 days at room temperature with shaking.
  • Day 2:
    • Transfer cell culture to micro test tube, then centrifuge and pellet cells.
    • Resuspend cells, add lysozyme, and freeze to rupture cell membranes; then centrifuge bacterial lysate to pellet membranes and debris.
    • Add high-salt chromatography binding buffer to bacterial lysate.
    • Load bacterial lysate onto columns.
  • Day 3:
    • 1. GFP binds to chromatography matrix in high-salt buffer.
    • 2. Add medium-salt buffer to wash bacterial proteins from column.
    • 3. Add low-salt buffer to elute GFP.
    • Collect three fractions.
    • Separate GFP from bacterial proteins
    • Extension: Use protein gel electrophoresis to conduct quantitative and qualitative analysis of fractions
Purity Confirmation with Gel Electrophoresis
  • Vertical Electrophoresis: Used to check protein purity.
  • Process:
    • Running buffer, test tube fluids, and protein markers are run on an acrylic gel.
    • Electrical current separates fragments based on size.
    • Larger fragments move slower than smaller fragments.
  • Outcome:
    • If the protein is not pure, another attempt is needed.

Lesson 4.2: Organ Failure

  • Manufacturing of human proteins, including insulin, was discussed.
  • Insulin and Diabetes: Insulin regulates sugar levels in diabetics.
  • Diabetes Symptoms: Frequent urination, constant thirst, rapid weight loss.
  • Complications: Diabetic retinopathy, neuropathy, fatigue.
  • Kidney Failure: Unregulated diabetes can lead to kidney failure.
  • Kidney failure results in improper blood filtering and toxin buildup, leading to death if untreated.
  • Diana Jones' case illustrates the impact of long-term, poorly managed diabetes.
  • ESRD Diagnosis: Diana has end-stage renal disease (ESRD).
ESRD Diagnosis Tests:
  • Blood Urea Nitrogen Levels: 60 mg/dL
  • Blood Creatine Levels: 2.8 mg/dL
  • Blood Potassium Levels: 7.1 mEq/L
  • Red Blood Cell Count: 3.6 million cells/mcL
  • Glomerular Filtration Rate (GFR): 13 mL/min
  • Urinalysis: Presence of red and white blood cells, high albumin levels (300 mg/dL)
  • Blood Pressure: 140/90
  • EKG: Normal
  • GFR and urinalysis confirm ESRD.
Treatment Options for Kidney Failure

  • Kidney Transplant: Best option, but not always possible.

  • Dialysis: Artificial process to remove waste and excess water from the blood.

    • Hemodialysis:
      • Blood is filtered outside the body using a machine.
      • Blood is removed, filtered, and returned via an intravenous line.
    • Peritoneal Dialysis:
      • The peritoneum filters the blood and collects wastes.

  • Diana uses dialysis temporarily while awaiting a kidney transplant.

  • Family members are willing to donate a kidney.

Lesson 4.3: Transplant

  • Organ demand exceeds the supply of available organs.
Key Statistics
  • A name is added to the national transplant waiting list every 13 minutes.
  • More than 70 lives are saved daily by organ transplantation.
  • Almost 20 people die each day waiting for a donated organ.
  • Organ donation and allocation are regulated by federal guidelines.
  • Matching involves blood type and tissue type compatibility.
  • The average national wait time for a kidney is longer than three years.
  • More than 78,000 people in the United States are waiting for a kidney transplant.
  • Family members are preferred donors due to higher compatibility and shorter wait times.
Organ Allocation Policies
  • Organ allocation is guided by federal policies, including NOTA and OPTN.
  • NOTA (National Organ Transplant Act):
    • Outlaws the sale of human organs.
    • Specifies medical criteria for organ allocation (compatibility, medical urgency for heart, liver, and intestine transplants).
    • Excludes social criteria (celebrity status, wealth, prison status).
    • Allows HIV-positive individuals in an asymptomatic state to be considered for transplantation with informed consent.
  • OPTN (Organ Procurement and Transplantation Network) Allocates Organs based on:
    • Compatibility of the donor and recipient.
    • Geographical proximity between donor and recipient.
    • Time on a waiting list.
    • Age of recipient (preference given to children).
Testing for Organ Match
  • Blood Typing:
    • Identifies blood types (A, B, AB, O) and Rh factors (+ or -).
    • Blood is classified as positive or negative based on Rh factors.
    • Blood cells labeled “A+” contain both A antigens and Rh antigens. Someone who is “A-“ contains A antigens, but not Rh factor.
    • Incompatible blood types result in immediate disqualification.
    • Process: Mixing blood with anti-serum to detect agglutination (clumping).
    • Agglutination with a certain “anti-serum” means that whatever the serum was testing for is present.
  • HLA Typing:
    • Identifies Human Leukocyte Antigens (HLA) on white blood cells.
    • HLA antigens determine self/non-self recognition for the immune system.
    • These antigens are controlled by a set of genes on chromosome 6 called the Major Histocompatibility Complex (MHC).
    • Two Classes: Class I and Class II
      • Class I: HLA-A, HLA-B, and HLA-Cw
      • Class II: HLA-DR, HLA-DQ, and HLA-DP
        HLA typing involves testing for the presence of different versions of this gene.
      • Process: DNA is isolated, amplified with PCR, and sequenced to determine alleles.
  • Antibody Screening (Panel Reactive Antibody - PRA):
    • Assesses the recipient's HLA antibodies by mixing their serum with cells from 60 individuals.
    • Evaluates the likelihood of organ rejection.
    • Ideally, serum reactivity should be less than 50%.
  • Crossmatch Test:
    • Mixes donor white blood cells with recipient serum.
    • Determines if the recipient's body will attack the donor organ.
    • No agglutination is desired for a negative crossmatch (transplant can proceed).
Transplant Surgery
  • Two-step procedure: kidney removal from donor and implantation in recipient.
  • Kidney Removal (Donor):
    • Laparoscopic nephrectomy: minimally invasive surgery using trocars and a laparoscope.
    • Results in less pain, shorter hospital stays, and quicker recovery.
  • Kidney Implantation (Recipient):
    • The new kidney is attached to the iliac or femoral artery and the bladder.
    • The recipient receives a functional kidney and can avoid dialysis.
Transplant Careers
  • Anesthesiologist, transplant surgeon, surgical (perioperative) nurse, and pharmacist.

Lesson 4.4: Building a Better Body

  • Exploration of technologies to improve the human body.
Technologies Discussed:
  • Tissue Engineering: Replacement of damaged tissues/organs with lab-grown tissues.
  • Xenotransplantation: Transplantation of organs from one species to another.
    • Currently, valves from cows and pigs are used, and human skin and bladders are grown.
  • Bionics: Fusion of humans and machines.
    • Prosthetics and myoelectric arms.
    • Vision restoration for the blind using electrodes, cameras, and sensors.